The complete chloroplast genome of Illicium simonsii Maxim. (Illiciaceae), a species with important medicinal properties

Abstract Illicium simonsii Maxim (1888) is a medicinal species of the genus Illicium in the Illiciaceae family. It is commonly used to cure gastro-frigid vomiting, cystic hernia, gas pains in the chest, and scabies as folk medicine. To utilize its resources efficiently, the complete chloroplast genome of I. simonsii was sequenced, assembled, and annotated by using high-throughput sequencing data. The complete chloroplast genome was 143,038 bp in length, with a large single-copy region (LSC) of 101,094 bp, a short single-copy region (SSC) of 20,070 bp, and a pair of inverted repeats (IRs) of 21,874 bp. A total of 113 genes were annotated, including 79 protein-coding genes, 30 tRNA genes, and four rRNA genes. The phylogenetic tree exhibited that I. simonsii and Illicium burmanicum form a sister group, and were nested in the monophyletic clade of the Illicium genus.


Introduction
Illicium simonsii Maxim ( 1888) is an evergreen tree of the genus Illicium in Illiciaceae family, which is widely distributed in southwest China, northern Burma, and northeast India (Xia et al. 2008;Zhang et al. 2018).Most studies of I. simonsii have focused on phytochemistry, and revealed that it has significant value (Lin et al. 2007;Wu and Lin 2008;Liu et al. 2012;Guo et al. 2021).It is a poisonous plant because ansisatin (a toxic substance that causes convulsions, respiratory paralysis and death) was extracted from its leaves, flowers and fruit (Liu et al. 2012;Yang et al. 1991).However, it is a rich source of essential oils, sesquiterpenoids, lignans, and C 6 -C 3 compounds, and has been used in traditional Chinese medicine for the treatment of rheumatic arthritis and to relieve pain (Li et al. 2022;Li et al. 2020;Zhang et al. 2019).In reality, it is difficult to reliably identify I. simonsii using morphologies due to the high similarity in morphological characteristics of Illicium species.This greatly impedes the effective development and usage of the resource of I. simonsii.Fortunately, the chloroplast genome has the potential to be an important source of genetic markers for species identification and evolution.(Lee et al. 2019).Therefore, it is urgent and necessary to assemble and sequence the complete chloroplast genome of I. simonsii, which may be crucial in solving the problem of accurate species identification and phylogenetic research.

Materials and methods
The sample of a single individual of I. simonsii was collected from Kunming Botanic Garden, Yunnan Province, China (102.71� E, 25.04 � N; Figure 1).The voucher specimen was stored at the Herbarium of Yunnan Normal University (https://life.ynnu.edu.cn;Jianlin Hang, hjlynnu@163.com)under the voucher number y29.
The total genomic DNA was extracted from the isolated chloroplasts using a modified CTAB method (Porebski et al. 1997) and stored in Huang's laboratory at Yunnan Normal University.According to the criterion, we fragmented the DNA and used Illumina Hiseq X Ten sequencer to construct the genomic library for Illumina paired-end (PE) sequencing.We obtained about 3.8 Gb raw data and assembled the chloroplast genome of I. simonsii with NOVOPlasty v2.7.2 (Dierckxsens et al. 2016).Genious Primer v2023.0.1 was used to build a read coverage depth map to verify the accuracy of genome assembly (Figure S1).Then, the whole chloroplast genome sequence was corrected using BWA software (Wang et al. 2018).The complete chloroplast genome of I. simonsii was annotated using Geneious v2010.1.1 (Kearse et al. 2012), with Illicium verum as a reference genome (Genbank Accession No. KY085896).The genes or other feature-encoding regions were identified by BLAT-based homology searches, by profile HMM searches for protein and rRNA coding genes and two de novo predictors for tRNA genes.Illicium henryi (Genbank Accession No. KY085910) is utilized as a model for modification and improvement.The genome map (Figure 2) and gene structures (Figure S2) of its  S1).To assess the divergence of the genome sequences, sliding windows with a window length of 600 bp and a step size of 200 bp were used to calculate nucleotide diversity.
The complete chloroplast genomes of 21 relative species were aligned using the program MAFFT v7.47 (Katoh and Standley 2013) to identify the phylogenetic position of  I. simonsii.Then IQ_TREE v1.6.10 software was used to construct the maximum likelihood phylogenetic tree with 10,000 replicates (Nguyen et al. 2015).The best-fit model according to the Bayesian information criterion (BIC) is TIM þ FþR2 (Kalyaanamoorthy et al. 2017).
The phylogenetic tree (Figure 4) exhibited that I. simonsii was closely related to I. burmanicum, and formed a monophyletic clade with the other 10 species of the Illicium genus by 100% bootstrap value.Moreover, the species from the genus Kadsura and Schisandra formed monophyletic clades, respectively, were sister to the genus Illicium.

Discussion and conclusion
In this study, the complete chloroplast genome of I. simonsii was sequenced, assembled and annotated.The total length is 14,308 bp, containing a large single-copy region (LSC) of 101,094 bp, a short single-copy region (SSC) of 20,070 bp, and a pair of inverted repeats (IRs) of 21,874 bp.The boundaries of the IR regions and the LSC and SSC regions of the complete chloroplast genomes of the 12 closely related species were compared (Figure S4), and found that fewer genes were observed to be duplicated in the genus Illicium due to the IR region contraction of more than 10 kb.This is consistent with the findings of a different study (Debra et al. 2007).Highly variable regions of the complete chloroplast genomes can be used to exploit markers to identify closely related species (Dong et al. 2014) and to provide abundant information for further phylogenetic study (Dong et al. 2012).In the genus Illicium, we have discovered five highly variable areas that could potentially serve as molecular markers in the future.This could facilitate more precise species identification and better resource usage of the genus Illiccium.The phylogenetic tree constructed based on the complete chloroplast genome showed that species in the genus Illicium formed a monophyly and were closely related to the Schisandraceae which include the genus Schisandra and Kadsura.This conclusion is in line with earlier phylogenetic research (Hu 1950;Li and Zheng 2018).
In summary, the complete chloroplast genome of I. simonsii could provide a valuable genomic resource for species identification and further phylogenetic studies of Illicium species.

Figure 1 .
Figure 1.Images of the species I. simonsii.Images courtesy of corresponding author Yuan Huang.Fruit with 8-13 follicles.The leaves are leathery, lanceolate to ellipsoid, and lack stipules.

Figure 2 .
Figure 2. The complete chloroplast genome map of I. simonsii.This genome map was created using the CPGview.The species name was shown in the left top corner.The map contains six tracks by default.The GC content along the genome was plotted on the fifth track.The genes were shown on the sixth track.The optional codon usage bias was displayed in the parenthesis after the gene name.Genes were color-coded by their functional classification and were shown in the bottom left corner.The transcription directions for the inner and outer genes were clockwise and anticlockwise, respectively.

Figure 3 .
Figure 3. Results of sliding window analyses of 12 complete chloroplast genome sequences of the illicium species by using DnaSP 5.10.Five highly variation region were labeled individually with number.